Compositions and methods that modulate cholesterol in a companion animal

ABSTRACT

Cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal can be improved by adjusting the diet of the animal to increase the amount of a compound which positively or negatively modulates the cholesterol, high-density lipoproteins, or low-density lipoproteins or adjusting the diet of the animal to decrease the amount of a compound which positively or negatively modulates cholesterol, high-density lipoproteins, or low-density lipoproteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser. No. 62/434,553 filed Dec. 15, 2016, the disclosure of which is incorporated in its entirety herein by this reference.

BACKGROUND

Animals, particularly companion animals (e.g., dogs and cats), may develop hypercholesterolemia, an abnormal condition characterized by elevated serum cholesterol levels. Hypercholesterolemia may occur together with chronic renal failure, hypothyroidism, cholestasis, hyperadrenocorticoidism, diabetes mellitus, idiopathic hypercholesterolemia, lipoprotein lipase deficiency, and primary hyperchylomicronemia, for example.

Hypercholesterolemia is one of the major causes of atherosclerosis or coronary heart diseases. Hyperlipidemia is characterized by abnormally elevated levels of any or all lipids and/or lipoproteins in the blood. Disorders associated with hyperlipidemia and/or hypercholesterolemia include cardiovascular disorders. Cardiovascular disorders are a group of disorders of the heart and blood vessels and include coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, deep vein thrombosis and pulmonary embolism, etc.

Accordingly, it is beneficial to be able to lower the serum cholesterol levels.

SUMMARY

The present disclosure relates generally to pet food compositions; methods of enhancing nutritional benefit of a pet food; and methods for modulating cholesterol, high-density lipoproteins, and/or low-density lipoproteins in a companion animal. Specifically, the present disclosure relates to metabolites for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal.

The present inventors have developed a predictive model of cholesterol, high-density lipoproteins, and low-density lipoproteins by identifying metabolite compounds which correlate to cholesterol, high-density lipoproteins, or low-density lipoproteins. A very controlled study was employed to minimize other external factors by using multiple canines all fed the same diet. A validation model was then developed by feeding different levels of the identified compounds (via a dietary change) to a group of canines and measuring changes in the corresponding cholesterol/lipid parameter.

Accordingly, in a general embodiment, the present disclosure provides a pet food composition comprising: protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal. In one embodiment, the pet food composition can provide at least a 5% increase or a 5% decrease in the amount of cholesterol, high-density lipoproteins, or low-density lipoproteins present in the companion animal. In one aspect, the pet food can provide an increase. In another aspect, the pet food can comprise a decrease.

The present disclosure also provides methods of minimizing costs associated with production of a pet food; methods of enhancing nutritional benefit of a pet food; and methods for modulating the amount of cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal.

An advantage of one or more embodiments provided by the present disclosure is to increase or decrease cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal by adjusting the diet of the animal to increase the amount of a compound which positively modulates the specific cholesterol/lipoprotein or to decrease the amount of a compound which negatively modulates the specific cholesterol/lipoprotein.

Additional features and advantages are described herein and will be apparent from the following detailed description.

DETAILED DESCRIPTION Definitions

As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” or “the composition” includes two or more compositions. The term “and/or” used in the context of “X and/or Y” should be interpreted as “X,” or “Y,” or “X and Y.” Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative, and are not exclusive or comprehensive.

As used herein, “about” is understood to refer to numbers in a range of numerals, for example the range of −10% to +10% of the referenced number, within −5% to +5% of the referenced number, or in one aspect, within −1% to +1% of the referenced number, and in a specific aspect, within −0.1% to +0.1% of the referenced number. Furthermore, all numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All percentages expressed herein are by weight of the total weight of the food composition unless expressed otherwise. When reference is made to the pH, values correspond to pH measured at 25° C. with standard equipment. An “amount” can be the total amount of the referenced component per serving of the composition or per distinct unit of the composition and/or can be the weight percentage of the referenced component by dry weight. Moreover, an “amount” includes zero; for example, the recitation of an amount of a compound does not necessarily mean that the compound is present, unless followed by a range that excludes zero.

The terms “food,” “food product” and “food composition” mean a product or composition that is intended for ingestion by an animal, including a human, and provides at least one nutrient to the animal. Further in this regard, these terms mean that the product or composition is in a form ready for consumption and is not merely an intermediate from which a consumable product or composition is made, although other food compositions can be added in some embodiments. The term “pet food” means any food composition intended to be consumed by a pet. The term “pet” means any animal which could benefit from or enjoy the compositions provided by the present disclosure. For example, the pet can be an avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal, but the pet can be any suitable animal.

The term “companion animal” means a dog or a cat. In an embodiment, the compositions and methods disclosed herein involve a senior dog or a senior cat. Dogs are considered senior in the last 25% of their lives. The life span of a dog depends on its size and/or its breed, but for the present disclosure a senior dog is a dog that is at least 5 years of age (e.g., at least 6 years of age, at least 7 years of age, or at least 8 years of age). The life span of a cat also depends on its size and/or its breed, but for the present disclosure a senior cat is a cat that is at least 7 years of age (e.g., at least 8 years of age, at least 9 years of age, or at least 10 years of age).

As used herein, “metabolite” refers to a compound having biological activity in a companion animal that is an intermediate or product of metabolism, and includes precursors thereof. As used herein, “precursor” refers to any compound that metabolizes to a metabolite during metabolism in a companion animal. For example, if the specific metabolite is cysteine, “the metabolite” comprises a cysteine precursor (e.g., methionine).

As used herein, “high-density lipoproteins” refers to a type of lipoprotein that transports fats and cholesterol from cells, including within artery wall atheroma, to the liver for excretion or re-utilization.

As used herein, “low-density lipoproteins” refers to a type of lipoprotein that transports fats and cholesterol to cells, and are associated with increasing rates of accumulation of atherosclerosis within the walls of arteries over time.

As used herein, “cholesterol” refers to a type of lipid molecule that is biosynthesized by all animal cells and is an essential structural component of the cell membrane. Cholesterol has the following chemical structure:

The term “effective amount” means an amount of a compound of the present invention that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In one embodiment, the present metabolite or combination of metabolites can be present in an effective amount for modulating cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal.

The dosages expressed herein are in milligrams per kilogram of body weight per day (mg/kg/day) unless expressed otherwise.

The term “long-term administration” means periods of repeated administration or consumption in excess of one month. Periods of longer than two, three, or four months can be used for certain embodiments. Also, more extended periods can be used that include longer than 5, 6, 7, 8, 9, or 10 months. Periods in excess of 11 months or 1 year can also be used. Longer term use extending over 1, 2, 3, or more years are included in the invention. For certain aging animals, the animal will continue consuming on a regular basis for the remainder of its life. This can also be referred to as consumption for “extended” periods.

The term “regular basis” or “regular administration” means at least monthly dosing with the compositions or consumption of the compositions, and in one aspect, means at least weekly dosing. More frequent dosing or consumption, such as twice or three times weekly, can be performed in certain embodiments. Still, in other embodiments, regimens can be used that comprise at least once daily consumption. The skilled artisan will appreciate that the blood level of a compound or certain metabolites of that compound or which result after the consumption of that compound, may be a useful tool for assessing or determining dosing frequency. For example, for determining feeding amounts for pet food compositions comprising a certain metabolite, the blood concentration of that metabolite, may provide useful information. A frequency, regardless of whether expressly exemplified herein, that allows maintenance of a desired blood level of the measured compound, such as a metabolite, within acceptable ranges can be useful herein. The skilled artisan will appreciate that feeding amounts will be a function of the composition that is being consumed or administered as well as the animal consuming the food, and some food compositions may require more or less frequent administration to maintain a desired blood level of the measured compound (e.g., a metabolite).

The relative terms “improve,” “increase,” “enhance,” “decrease” and the like refer to the effects of the composition disclosed herein (a composition comprising a metabolites) relative to a composition having a lower amount or lacking such metabolites, but otherwise identical.

A “blended” composition merely has at least two components having at least one different characteristic relative to each other. In one aspect, moisture content and water activity can be different in the context of the present disclosure. In this regard, description of a composition as “blended” does not imply that the blended composition has been subjected to processing sometimes referenced as “blending,” namely mixing components so that they are indistinguishable from each other, and, in one aspect, such processing is avoided when mixing one component with the other components to form a blended composition (e.g., mixing a dry component with a wet or semi-moist component). Further in this regard, in a blended composition each of the at least two components having at least one different characteristic relative to each other can retain their distinct identity and appearance.

“Wet food” means a pet food having a moisture content from about 50% to about 90%, and in one aspect, from about 70% to about 90%. “Dry food” means a pet food having a moisture content less than about 20%, and in one aspect, less than about 15%, and in a specific aspect, less than about 10%. “Semi-moist food” means a pet food having a moisture content from about 20% to about 50%, and in one aspect, from about 25% to about 35%.

“Kibbles” is used synonymously with “chunks” herein and both terms mean pieces of dry or semi-moist pet food which can have a pellet shape or any other shape and can be made by slicing a food composition into separate pieces. Non-limiting examples of kibbles include particulates; pellets; pieces of pet food, dehydrated meat, meat analog, vegetables, and combinations thereof; and pet snacks, such as meat or vegetable jerky, rawhide, and biscuits. A “meat analog” is a meat emulsion product that resembles pieces of natural meat in appearance, texture, and physical structure.

The term “dietary supplement” means a product that is intended to be ingested in addition to the normal animal diet. Dietary supplements may be in any form, e.g., solid, liquid, gel, tablets, capsules, powder, and the like. In one aspect, they can be provided in convenient dosage forms. In some embodiments, they can be provided in bulk consumer packages such as bulk powders, liquids, gels, or oils. In other embodiments, supplements can be provided in bulk quantities to be included in other food items such as snacks, treats, supplement bars, beverages and the like.

The compositions disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of” and “consisting of” the components identified. Similarly, the methods disclosed herein may lack any step that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of” and “consisting of” the steps identified. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein unless explicitly and directly stated otherwise.

The present discussion of embodiments, aspects, examples, etc. are independent in that they can apply to all methods and compositions. For example, a metabolite used in a pet food composition can also be used in the method of modulating or a method of minimizing costs associated with making such a pet food, and vice versa.

Embodiments

In an aspect of the present disclosure, a pet food composition can comprise protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal. In one aspect, the pet food composition can include at least 6 distinct metabolites for individually modulating each one of cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal. In one embodiment, the pet food composition can provide a decrease in at least one of cholesterol and low-density lipoproteins in the companion animal. In another embodiment, the pet food composition can provide an increase in high-density lipoproteins in the companion animal. In one aspect, the companion animal can be a senior dog or a senior cat.

In another aspect of the present disclosure, a method of modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal in a companion animal is provided. The method comprises administering to the companion animal a pet food composition comprising protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in the companion animal. In one aspect, the companion animal can be a senior dog or a senior cat.

Yet another aspect of the present disclosure is a method of minimizing costs associated with production of a pet food having a first formulation designed for consumption by a companion animal, such as a senior dog or a senior cat. A further aspect of the present disclosure is a method of enhancing nutritional benefit of a pet food having a first formulation designed for consumption by a companion animal, such as a senior dog or a senior cat. These methods comprise adjusting the first formulation of the pet food to be a second formulation. At least one of the first and second formulations comprises a metabolite for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in the companion animal. The adjusting comprises changing an amount of the metabolite in the first formulation to a different amount in the second formulation.

“Minimizing” costs means that the costs associated with making the second formulation are less than the costs associated with making the first formulation, for example on a per serving basis, per unit weight, per unit volume, per total energy, and the like. “Enhanced” nutritional benefit means that the nutritional benefit of the second formulation is greater than the nutritional benefit of the first formulation.

As discussed herein, the pet food compositions and methods can contain a metabolite or multiple metabolites for modulating cholesterol, high-density lipoproteins, or low-density lipoproteins. In various aspects, the compositions and methods can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 metabolites for any one of the cholesterol, high-density lipoproteins, or low-density lipoproteins, or multiple parameters of cholesterol, high-density lipoproteins, or low-density lipoproteins, or for each cholesterol, high-density lipoproteins, and low-density lipoproteins. As such, in one embodiment, the composition can comprise at least 12 metabolites, including at least 2 metabolites for individually modulating each one of cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal. In another embodiment, the composition can comprise at least 18 metabolites, including at least 3 metabolites for individually modulating each one of cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal. Additionally, in one embodiment, the composition can comprise at least four distinct metabolites that modulate at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins. Further, in one embodiment, the composition can comprise at least five distinct metabolites that modulate at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins. Still, in another embodiment, the method can modulate at least two parameters selected from the group consisting of cholesterol, high-density lipoproteins, and low-density lipoproteins and where the composition comprises at least six distinct metabolites, three distinct metabolites for modulating each of the at least two parameters. Yet, in another embodiment, the method can modulate each one of cholesterol, high-density lipoproteins, and low-density lipoproteins and where the composition comprises at least nine distinct metabolites, three distinct metabolites for modulating each of the at least three parameters. As previously noted, the metabolite and cholesterol/lipoprotein relationships can be used for both method and composition embodiments.

In some embodiments, the metabolite negatively modulates the specific cholesterol/lipoprotein, and the changing of the amount of the metabolite comprises decreasing the amount of the metabolite. In other embodiments, the metabolite negatively modulates the specific cholesterol/lipoprotein, and the changing of the amount of the metabolite comprises increasing the amount of the metabolite. In some embodiments, the metabolite positively modulates the specific cholesterol/lipoprotein, and the changing of the amount of the metabolite comprises increasing the amount of the metabolite. In other embodiments, the metabolite positively modulates the specific cholesterol/lipoprotein, and the changing of the amount of the metabolite comprises decreasing the amount of the metabolite. These are not mutually exclusive embodiments; a particular embodiment can comprise decreasing the amount of a metabolite that negatively modulates a first specific cholesterol/lipoprotein and increasing the amount of a metabolite that positively modulates a second specific cholesterol/lipoprotein, and the first and second specific cholesterol/lipoprotein can be the same or different specific cholesterol/lipoprotein (e.g., one or more of cholesterol, high-density lipoproteins, or low-density lipoproteins).

Decreasing the amount of the metabolite can comprise decreasing the amount of the metabolite directly and/or decreasing the amount of an ingredient which comprises the metabolite. In some embodiments, decreasing the amount of the metabolite can comprise decreasing the amount of a precursor of the metabolite directly and/or decreasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, less of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

Increasing the amount of the metabolite can comprise increasing the amount of the metabolite directly and/or increasing the amount of an ingredient which comprises the metabolite. In some embodiments, increasing the amount of the metabolite can comprise increasing the amount of a precursor of the metabolite directly and/or increasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, more of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

Generally, the methods and compositions described herein can provide an increase or a decrease in at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in the companion animal. In some embodiment, the increase or decrease can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50%. In one aspect, the compositions and methods can provide an increase. In another aspect, the compositions and methods can provide a decrease. In another aspect, the compositions and methods can provide an increase in one parameter and a decrease in another parameter. As such, the present modulation, and resulting increase or decrease, can provide treatment of one of the following: hypercholesterolemia, hyperlipidemia, coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, deep vein thrombosis, inflammation or pulmonary embolism.

In one embodiment, the increase or decrease can be for at least two of cholesterol, high-density lipoproteins, and low-density lipoproteins. In another embodiment, the increase or decrease can be each one of cholesterol, high-density lipoproteins, and low-density lipoproteins.

Ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

In each of these compositions and methods, the pet food composition can be a wet food, a semi-moist food or a dry food. In an embodiment, the pet food composition is one or more components of a blended composition. In some embodiments, the pet food composition is a kibble, and in some embodiments, the pet food composition is a meat analog. Additionally, in another embodiment, the present composition for modulating cholesterol, high-density lipoproteins, and low-density lipoproteins can be a dietary supplement comprising the metabolites described herein. Further, a method of modulating cholesterol, high-density lipoproteins, or low-density lipoproteins can include administering to the companion animal the dietary supplement.

Such pet food compositions can be administered to the companion animal in amounts ranging from about 3 g of pet food per 1 lb body weight to about 16 g of pet food per 1 lb body weight of the companion animal. Additionally, the metabolites can be present in amounts from about 0.01 weight % to about 10 weight % of the food composition. In one aspect, the metabolites can be present in concentrations of about 0.01 to about 1,000 mg/kg of food. In another aspect, the metabolites can be present in concentrations from about 1 IU to about 500,000 IU per kilogram of food. In one embodiment, the pet food composition can be administered to the companion animal in amounts sufficient to maintain the health and/or body weight of the animal. In one aspect, the administration can be regular administration.

As noted above and detailed later in this application, the present inventors identified metabolite compounds which correlate to cholesterol, high-density lipoproteins, or low-density lipoproteins. Thus, the metabolite in the pet food composition can be one of these compounds. Nevertheless, the metabolite can be any metabolite for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal, even if the metabolite is not explicitly disclosed herein. For example, the metabolite can be a compound identified using the methods disclosed herein but not itself explicitly disclosed herein. Furthermore, the metabolite can be a compound identified using a method not disclosed herein if the compound is reliably correlated to at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins.

As a non-limiting example, the metabolite can modulate cholesterol and can be selected from the group consisting of palmitoyl sphingomyelin, X-13215, 1-stearoylglycerol (1-monostearin), alpha-tocopherol, 7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 7-beta-hydroxycholesterol, X-17010, glycerophosphorylcholine (GPC), X-16120, 2-linoleoylglycerophosphoethanolamine, 5-methyl-2′-deoxycytidine, caproate (6:0), 1-linoleoylglycerophosphoethanolamine, glycerol 3-phosphate (G3P), 2-arachidonoylglycerophosphoethanolamine, glycerol 2-phosphate, dihydrocholesterol, pelargonate (9:0), stearamide, 2-palmitoylglycerophosphoethanolamine, malate, heptanoate (7:0), and mixtures thereof. In one embodiment, the metabolite can modulate cholesterol and can be selected from the group consisting of palmitoyl sphingomyelin, 1-stearoylglycerol (1-monostearin), alpha-tocopherol, 7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 7-beta-hydroxycholesterol, glycerophosphorylcholine (GPC), 2-linoleoylglycerophosphoethanolamine, 5-methyl-2′-deoxycytidine, caproate (6:0), 1-linoleoylglycerophosphoethanolamine, glycerol 3-phosphate (G3P), 2-arachidonoylglycerophosphoethanolamine, glycerol 2-phosphate, dihydrocholesterol, pelargonate (9:0), stearamide, 2-palmitoylglycerophosphoethanolamine, malate, heptanoate (7:0), and mixtures thereof.

As another non-limiting example, the metabolite can modulate high-density lipoproteins and can be selected from the group consisting of palmitoyl sphingomyelin, X-13215, alpha-tocopherol, 1-stearoylglycerol (1-monostearin), 7-alpha-hydroxycholesterol, X-17010, 1-arachidonoylglycerophosphocholine, 2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate (20:4n6), X-17612, 3-hydroxybutyrate (BHBA), 2-arachidonoylglycerophosphocholine, pelargonate (9:0), stearamide, N2,N2-dimethylguanosine, 1-linoleoylglycerophosphocholine, dihydrocholesterol, X-11412, 1-stearoylglycerophosphocholine, X-17147, glycerate, heptanoate (7:0), 1-docosahexaenoylglycerophosphocholine, dihomo-linoleate (20:2n6), 2-linoleoylglycerophosphocholine, 2-palmitoylglycerophosphocholine, and mixtures thereof. In one embodiment, the metabolite can modulate high-density lipoproteins and can be selected from the group consisting of palmitoyl sphingomyelin, alpha-tocopherol, 1-stearoylglycerol (1-monostearin), 7-alpha-hydroxycholesterol, 1-arachidonoylglycerophospho choline, 2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate (20:4n6), 3-hydroxybutyrate (BHBA), 2-arachidonoylglycerophosphocholine, pelargonate (9:0), stearamide, N2,N2-dimethylguanosine, 1-linoleoylglycerophosphocholine, dihydrocholesterol, 1-stearoylglycerophosphocholine, glycerate, heptanoate (7:0), 1-docosahexaenoylglycerophosphocholine, dihomo-linoleate (20:2n6), 2-linoleoylglycerophosphocholine, 2-palmitoylglycerophosphocholine, and mixtures thereof.

As yet another non-limiting example, the metabolite can modulate low-density lipoproteins and can be selected from the group consisting of 1-stearoylglycerol (1-monostearin), X-13215, palmitoyl sphingomyelin, alpha-tocopherol, 7-alpha-hydroxycholesterol, 2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC), 7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine, X-17010, 2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol (1-monopalmitin), caproate (6:0), 5-methyl-2′-deoxycytidine, 2-arachidonoylglycerophosphoethanolamine, malate, glycerol 2-phosphate, glycerol 3-phosphate (G3P), 2-oleoylglycerophosphoethanolamine, N2,N2-dimethylguanosine, and mixtures thereof. In one embodiment, the metabolite can modulate low-density lipoproteins and can be selected from the group consisting of 1-stearoylglycerol (1-monostearin), palmitoyl sphingomyelin, alpha-tocopherol, 7-alpha-hydroxycholesterol, 2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC), 7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine, 2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol (1-monopalmitin), caproate (6:0), 5-methyl-2′-deoxycytidine, 2-arachidonoylglycerophosphoethanolamine, malate, glycerol 2-phosphate, glycerol 3-phosphate (G3P), 2-oleoylglycerophosphoethanolamine, N2,N2-dimethylguanosine, and mixtures thereof.

In yet another aspect of the present disclosure, a method enhances nutritional benefit of a pet food having a first formulation designed for consumption by companion animals, and the method comprises administering the pet food having the first formulation to a first companion animal. The method further comprises measuring in a sample of body fluid from the companion animal (e.g., plasma) an amount of a surrogate marker comprising a metabolite that modulates at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins. The method further comprises adjusting the first formulation of the pet food to be a second formulation having a difference from the first formulation selected from the group consisting of (i) an ingredient is present in the second formulation and is absent in the first formulation, (ii) an ingredient is absent in the second formulation and is present in the first formulation, (iii) an ingredient is present in the first and second formulations but in a different amount, and (iv) combinations thereof. The adjusting is based at least partially on the amount of the surrogate marker measured in the previous step.

The adjusting can comprise directly decreasing the amount of a metabolite negatively modulating a specific cholesterol/lipoprotein and/or decreasing the amount of an ingredient which comprises a metabolite negatively modulating a specific cholesterol/lipoprotein. In some embodiments, decreasing the amount of the metabolite can comprise decreasing the amount of a precursor of the metabolite directly and/or decreasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, less of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

The adjusting can comprise increasing the amount of the metabolite can comprise directly increasing the amount of a metabolite positively modulating a specific cholesterol/lipoprotein and/or increasing the amount of an ingredient which comprises a metabolite positively modulating a specific cholesterol/lipoprotein. In some embodiments, increasing the amount of the metabolite can comprise increasing the amount of a precursor of the metabolite directly and/or increasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, more of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

As noted above, ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

The method further comprises producing the pet food in the second formulation. In an embodiment, the method comprises administering the pet food having the second formulation to a second companion animal.

This method can be used to provide customized nutrition for a specific companion animal. For example, the first and second companion animal can be the same specific companion animal such that the animal who is administered the pet food having the first formulation has one or more of their specific cholesterol/lipoprotein of the first formulation assessed. Then this same animal is provided with the resultant second formulation which this animal will more effectively modulate with respect to at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins. Consequently, a pet owner can compensate for their pet's age-induced changes in one or more of cholesterol, high-density lipoproteins, or low-density lipoproteins.

Alternatively or additionally, this method can be used to provide customized nutrition for companion animals who share one or more of a gender, an approximate age, an approximate size (e.g., body weight, height, and/or length) or a breed. For example, the second companion animal can be a different specific animal than the first companion animal but has a characteristic selected from the group consisting of (i) about the same age as the first companion animal, (ii) about the same size as the first companion animal, (iii) the same breed as the first companion animal, (iv) the same gender as the first companion animal, and (iv) combinations thereof. In one embodiment, the second companion animal can be one of a plurality of companion animals who each share the characteristic with the first companion animal. The method can comprise administering the pet food having the second formulation to the plurality of companion animals. In an embodiment, at least a portion of the plurality of companion animals is located remotely relative to the first companion animal.

The pet food compositions disclosed herein can be any food formulated for consumption by a pet such as a companion animal. In an embodiment, the pet food composition provides complete nutrition as defined by the Association of American Feed Control Officials (AAFCO) and which depends on the type of animal for which the composition is intended (e.g., dog or cat).

The pet food composition can comprise meat, such as emulsified meat. Examples of suitable meat include poultry, beef, pork, lamb and fish, especially those types of meats suitable for pets. The meat can include any additional parts of an animal including offal. Some or all of the meat can be provided as one or more meat meals, namely meat that has been dried and ground to form substantially uniform-sized particles and as defined by AAFCO. Additionally or alternatively, vegetable protein can be used, such as pea protein, corn protein (e.g., ground corn or corn gluten), wheat protein (e.g., ground wheat or wheat gluten), soy protein (e.g., soybean meal, soy concentrate, or soy isolate), rice protein (e.g., ground rice or rice gluten) and the like.

The pet food compositions disclosed herein can comprise vegetable oil, a flavorant, a colorant and water. Suitable vegetable oils include soybean oil, corn oil, cottonseed oil, sunflower oil, canola oil, peanut oil, safflower oil, and the like. Examples of suitable flavorants include yeast, tallow, rendered animal meals (e.g., poultry, beef, lamb, pork), flavor extracts or blends (e.g., grilled beef), animal digests, and the like. Suitable colorants include FD&C colors, such as blue no. 1, blue no. 2, green no. 3, red no. 3, red no. 40, yellow no. 5, yellow no. 6, and the like; natural colors, such as caramel coloring, annatto, chlorophyllin, cochineal, betanin, turmeric, saffron, paprika, lycopene, elderberry juice, pandan, butterfly pea and the like; titanium dioxide; and any suitable food colorant known to the skilled artisan.

The pet food compositions disclosed herein can optionally include additional ingredients, such as other grains and/or other starches additionally or alternatively to flour, amino acids, fibers, sugars, animal oils, aromas, other oils additionally or alternatively to vegetable oil, humectants, preservatives, polyols, salts, oral care ingredients, antioxidants, vitamins, minerals, probiotic microorganisms, bioactive molecules or combinations thereof.

Suitable starches include a grain such as corn, rice, wheat, barley, oats, soy and the like, and mixtures of these grains, and can be included at least partially in any flour. Suitable humectants include salt, sugars, propylene glycol and polyhydric glycols such as glycerin and sorbitol, and the like. Suitable oral care ingredients include alfalfa nutrient concentrate containing chlorophyll, sodium bicarbonate, phosphates (e.g., tricalcium phosphate, acid pyrophosphates, tetrasodium pyrophosphate, metaphosphates, and orthophosphates), peppermint, cloves, parsley, ginger and the like. Examples of suitable antioxidants include butylated hydroxyanisole (“BHA”) and butylated hydroxytoluene (“BHT”), vitamin E (tocopherols), and the like.

Non-limiting examples of vitamins that can be used include Vitamins A, B-complex (such as B-1, B-2, B-6 and B-12), C, D, E and K, niacin and acid vitamins such as pantothenic acid and folic acid and biotin. Non-limiting examples of suitable minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium, boron and the like.

-   -   n-limiting examples of suitable preservatives include potassium         sorbate, sorbic acid, sodium methyl para-hydroxybenzoate,         calcium propionate, propionic acid, and combinations thereof.

Specific amounts for each additional ingredient in the pet food compositions disclosed herein will depend on a variety of factors such as the ingredient included in the first edible material and any second edible material; the species of animal; the animal's age, body weight, general health, sex, and diet; the animal's consumption rate; the purpose for which the food product is administered to the animal; and the like. Therefore, the components and their amounts may vary widely.

For example, the amount of any of the above-noted ingredients can be decreased or increased based on the estimated effect on one or more of cholesterol, high-density lipoproteins, or low-density lipoproteins (e.g., an effect identified by one of the methods disclosed herein). In an embodiment, the amount of one or more of the above-noted ingredients can be increased if such ingredients comprise a metabolite that positively modulates one or more of cholesterol, high-density lipoproteins, or low-density lipoproteins. Additionally or alternatively, the amount of one or more of the above-noted ingredients can be decreased if such ingredients comprise a metabolite that negatively modulates one or more of cholesterol, high-density lipoproteins, or low-density lipoproteins.

As noted above, ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

Examples

The following non-limiting examples are illustrative of embodiments of the present disclosure.

Methods

Each of the examples was derived from the following study.

83 Canines were all fed Diet A for 5 weeks, followed by a 1 week transition period and then 15 were fed Diet B for 5 weeks as shown in the Table 1 below. Plasma samples were taken after overnight fasting using EDTA vacutainer tubes during the fifth week of feeding of each diet. After centrifugation, plasma was aliquoted into cryovials and frozen at −80° C. Cholesterol, high-density lipoproteins, and low-density lipoproteins were measured using the Cobas® c311 or e411 clinical chemistry analyzer, according to manufacturer's directions.

TABLE 1 Moisture Basis Moisture % DM % Protein % Fat % Ash % Fiber % CHO % GE kcal/g Diet A As-Is 8.1 91.9 22.7 13.3 6.1 2.0 47.9 4.5 Dry 0 100 24.7 14.5 6.6 2.1 52.1 4.9 matter Diet B As-Is 76 24 9.1 10.5 1.8 0 2.6 1.7 Dry 0 100 38 43.7 7.5 0 10.8 6.9 Matter

Metabolomic analysis was carried out using the following methods by Metabolon Inc. Samples were extracted and split into equal parts for analysis on GC/MS and LC/MS/MS platforms. Proprietary software was used to match ions to an in-house library of standards for metabolite identification and for metabolite quantitation by peak area integration by Metabolon Inc. Mass and retention index are provided in the following tables such that each metabolite can be uniquely identified and individually distinguished.

At the time of analysis, samples were thawed and extracts prepared to remove protein, dislodge small molecules bound to protein or physically trapped in the precipitated protein matrix, and recover a wide range of chemically diverse metabolites. A separate aliquot of each experimental plasma sample was taken then pooled for the creation of “Client Matrix” (CMTRX) samples. These CMTRX samples were injected throughout the platform run and served as technical replicates, allowing variability in the quantitation of all consistently detected biochemicals to be determined and overall process variability and platform performance to be monitored. Extracts of all experimental and CMTRX samples were split for analysis on the GC/MS and LC/MS/MS platforms.

The CMTRX technical replicate samples were treated independently throughout the process as if they were client study samples. All process samples (CMTRX and Grob test mixtures of organic components used to assess GC column performance, process blanks, etc.) were spaced evenly among the injections for each day and all client samples were randomly distributed throughout each day's run. Data were collected over multiple platform run days and thus ‘block normalized’ by calculating the median values for each run-day block for each individual compound. This normalization minimizes any inter-day instrument gain or drift, but does not interfere with intra-day sample variability. Missing values (if any) were assumed to be below the level of detection for that biochemical with the instrumentation used and were imputed with the observed minimum for that particular biochemical.

A number of internal standards were added to each experimental and process standard sample just prior to injection into the mass spectrometers. A measure of the platform variability (7%) was determined by calculating the median relative standard deviation (RSD) for these internal standards. Because these standards are added to the samples immediately prior to injection into the instrument, this value reflects instrument variation. In addition, the median relative standard deviation (RSD) for the biochemicals that were consistently measured in the CMTRX represents the total variability within the process for the actual experimental samples and the variability in quantitation of the endogenous metabolites within these samples (12%). Results for the CMTRX and internal standards indicated that the platform produced data that met process specifications.

589 total metabolites were detected in plasma. This total corresponds to many biochemicals (401) that matched a named structure in the reference library (named compounds). The remaining biochemicals (188) represent distinct chemical entities (that is, they represent a single molecule of discrete molecular formula and structure), but they do not currently match a named biochemical in the reference library (unnamed/unknown compounds).

Example 1 (Cholesterol)

Metabolite correlations with cholesterol were determined based on plasma metabolomics (Table 2). This provided a predictive model of compounds which can influence cholesterol either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 3), and noting changes in cholesterol (Table 4), served as a validation model. The metabolite compositions of the two different diets were determined to identify relative levels of specific compounds. Those validated by the model are shown in Table 5.

TABLE 2 Metabolite correlations with cholesterol. Correlations with a P value < 0.01 are reported. Correlation Retention ID Correlation P-Value Index Mass palmitoyl sphingomyelin 0.7526 0.00E+00 2524 311.3 X - 13215 0.7468 0.00E+00 5179 149.2 1-stearoylglycerol (1-monostearin) 0.7003 1.11E−15 2186.6 399.4 alpha-tocopherol 0.6558 2.32E−13 2305.4 502.5 stearoyl sphingomyelin 0.6081 3.13E−11 #N/A #N/A 7-alpha-hydroxycholesterol 0.523 3.29E−08 2300 456.2 2-ethylhexanoate 0.4816 5.17E−07 1210.5 201.2 1-palmitoylglycerol (1-monopalmitin) 0.4607 1.81E−06 2119.5 371.3 dimethylarginine (SDMA + ADMA) −0.455 2.51E−06 812 203.2 7-beta-hydroxycholesterol 0.4381 6.43E−06 2340 456.4 X - 17010 0.4193 1.73E−05 3169.5 189 glycerophosphorylcholine (GPC) 0.4134 2.33E−05 694 258.1 X - 16120 0.4021 4.06E−05 1271.7 164.1 lathosterol 0.3998 4.53E−05 2337 255.2 2-linoleoylglycerophosphoethanolamine 0.3768 0.0001 5650 476.4 S-propylcysteine −0.3822 0.0001 2000.5 164.1 5-methyl-2′-deoxycytidine −0.3627 0.0002 1480 242 caproate (6:0) 0.3678 0.0002 2766 115.2 X - 11987 −0.3694 0.0002 1409 139.2 threonine 0.3611 0.0003 713 120.1 lysine 0.3529 0.0004 1836.7 317.2 X - 12355 −0.349 0.0004 3559 206.1 1-linoleoylglycerophosphoethanolamine 0.3451 0.0005 5725 476.3 threonate 0.337 0.0007 1560.7 292.1 glycerol 3-phosphate (G3P) 0.3318 0.0008 1719.7 357.1 arabinose 0.3229 0.0012 1631.6 217 N2,N2-dimethylguanosine −0.3207 0.0013 2157 312.1 2-arachidonoylglycerophosphoethanolamine 0.3084 0.002 5674 500.3 glycerol 2-phosphate 0.3047 0.0023 1691.8 243 dihydrocholesterol 0.3033 0.0024 2320.8 215.2 pelargonate (9:0) 0.3008 0.0026 4847 157.2 caprylate (8:0) 0.298 0.0029 4367 143.2 stearamide 0.2949 0.0032 2071 131 2-palmitoylglycerophosphoethanolamine 0.294 0.0033 5790 452.3 X - 11437 −0.2938 0.0033 2888 231 X - 16940 −0.2931 0.0034 1694.1 204.9 X - 12029 0.2834 0.0047 683 329.1 N1-methyladenosine −0.2791 0.0054 #N/A #N/A alpha-ketoglutarate −0.276 0.0059 1779 419.1 4-vinylphenol sulfate −0.2711 0.0069 3323 199.1 X - 16945 −0.2702 0.0071 3457.9 351 thymol sulfate −0.2687 0.0075 4395 229.1 malate −0.2634 0.0088 1502 233 pyrophosphate (PPi) −0.2629 0.0089 1642.3 451 cystine 0.2605 0.0096 2015.3 145.9 heptanoate (7:0) 0.2599 0.0097 3695 129.2 X - 12668 −0.2599 0.0098 2318 246.1

TABLE 3 Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values (if any) were imputed with the observed minimum for that particular compound). ID A B palmitoyl sphingomyelin 0.03 0.2 X - 13215 0.02 0.1 1-stearoylglycerol (1-monostearin) 0.38 1.21 alpha-tocopherol 0.01 0.03 stearoyl sphingomyelin N/A N/A 7-alpha-hydroxycholesterol 0.74 4.91 2-ethylhexanoate 0.06 0.02 1-palmitoylglycerol (1-monopalmitin) 0.87 1.52 dimethylarginine (SDMA + ADMA) 0.29 0.63 7-beta-hydroxycholesterol 0.91 1.81 X - 17010 0.68 2.51 glycerophosphorylcholine (GPC) 57.82 106.56 X - 16120 0.45 0.45 lathosterol 0.41 0.41 2-linoleoylglycerophosphoethanolamine 0.56 2.58 S-propylcysteine 0.05 0.05 5-methyl-2′-deoxycytidine 1.05 0.61 caproate (6:0) 0.35 0.49 X - 11987 0.14 0.14 threonine 3.43 3.44 lysine 33.69 6.43 X - 12355 0.03 0.03 1-linoleoylglycerophosphoethanolamine 0.48 2.77 threonate 0.61 0.22 glycerol 3-phosphate (G3P) 3.12 10.24 arabinose 10.21 2.48 N2,N2-dimethylguanosine 0.59 0.54 2-arachidonoylglycerophosphoethanolamine 0.33 10.33 glycerol 2-phosphate 3.63 4.01 dihydrocholesterol 0.23 0.74 pelargonate (9:0) 0.07 0.1 caprylate (8:0) 0.1 0.12 stearamide 0.99 1.06 2-palmitoylglycerophosphoethanolamine 0.69 2.75 X - 11437 0.05 0.05 X - 16940 0.12 0.12 X - 12029 0.35 0.35 N1-methyladenosine #N/A #N/A alpha-ketoglutarate 0.22 0.43 4-vinylphenol sulfate 0.02 0.02 X - 16945 0.07 0.07 thymol sulfate 0.41 0.41 malate 9.52 4.85 pyrophosphate (PPi) 0.15 0.34 cystine 0.37 0.37 heptanoate (7:0) 0.27 0.39 X - 12668 0.13 0.13

TABLE 4 Cholesterol in response to diet A and Diet B. Difference Diet A Diet B p- Parameter Mean StdDev Mean StdDev Mean StdErr value Cholesterol 190 36.0 253.4 38.7 −63.4 5.2 0

TABLE 5 Compounds validated by dietary change. Retention ID Index Mass palmitoyl sphingomyelin 2524 311.3 X - 13215 5179 149.2 1-stearoylglycerol (1-monostearin) 2186.6 399.4 alpha-tocopherol 2305.4 502.5 7-alpha-hydroxycholesterol 2300 456.2 1-palmitoylglycerol (1-monopalmitin) 2119.5 371.3 7-beta-hydroxycholesterol 2340 456.4 X - 17010 3169.5 189 glycerophosphorylcholine (GPC) 694 258.1 X - 16120 1271.7 164.1 2-linoleoylglycerophosphoethanolamine 5650 476.4 5-methyl-2′-deoxycytidine 1480 242 caproate (6:0) 2766 115.2 1-linoleoylglycerophosphoethanolamine 5725 476.3 glycerol 3-phosphate (G3P) 1719.7 357.1 2-arachidonoylglycerophosphoethanolamine 5674 500.3 glycerol 2-phosphate 1691.8 243 dihydrocholesterol 2320.8 215.2 pelargonate (9:0) 4847 157.2 stearamide 2071 131 2-palmitoylglycerophosphoethanolamine 5790 452.3 malate 1502 233 heptanoate (7:0) 3695 129.2

Example 2 (High-Density Lipoprotein)

Metabolite correlations with high-density lipoproteins were determined based on plasma metabolomics (Table 6). This provided a predictive model of compounds which can influence high-density lipoproteins either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 7), and noting changes in high-density lipoproteins (Table 8), served as a validation model. The metabolite compositions of the two different diets were determined to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 9).

TABLE 6 Metabolite correlations with high-density lipoproteins. Correlations with a P value < 0.01 are reported. Correlation Retention ID Correlation P-Value Index Mass palmitoyl sphingomyelin 0.7578 0.00E+00 2524 311.3 stearoyl sphingomyelin 0.7318 0.00E+00 N/A N/A X - 13215 0.6493 4.78E−13 5179 149.2 alpha-tocopherol 0.6197 1.03E−11 2305.4 502.5 1-stearoylglycerol (1-monostearin) 0.5654 1.32E−09 2186.6 399.4 2-ethylhexanoate 0.4585 2.06E−06 1210.5 201.2 X - 16120 0.4562 2.35E−06 1271.7 164.1 7-alpha-hydroxycholesterol 0.4552 2.48E−06 2300 456.2 X - 17010 0.4338 8.11E−06 3169.5 189 dimethylarginine (SDMA + ADMA) −0.4303 9.76E−06 812 203.2 threonate 0.4123 2.45E−05 1560.7 292.1 S-propylcysteine −0.4033 3.83E−05 2000.5 164.1 X - 12355 −0.3914 6.75E−05 3559 206.1 1-arachidonoylglycerophosphocholine 0.375 0.0001 5554 544.3 2-docosapentaenoylglycerophosphocholine 0.3744 0.0001 5532 570.3 X - 11987 −0.3817 0.0001 1409 139.2 7-beta-hydroxycholesterol 0.3683 0.0002 2340 456.4 1-palmitoylglycerol (1-monopalmitin) 0.3598 0.0003 2119.5 371.3 X - 16940 −0.3518 0.0004 1694.1 204.9 caprylate (8:0) 0.3467 0.0005 4367 143.2 X - 11549 0.3379 0.0007 5093 339.3 1-docosapentaenoylglycerophosphocholine 0.3357 0.0007 5584 570.4 caproate (6:0) 0.3355 0.0007 2766 115.2 arachidonate (20:4n6) 0.3345 0.0008 5525 303.4 X - 17612 0.3344 0.0008 4298.5 567.4 3-hydroxybutyrate (BHBA) 0.3321 0.0008 1203.5 116.9 2-arachidonoylglycerophosphocholine 0.3236 0.0012 5524 544.3 2-stearoylglycerophosphocholine 0.3234 0.0012 5780 524.4 pelargonate (9:0) 0.3229 0.0012 4847 157.2 X - 15546 −0.3185 0.0014 1861 271.1 N-stearoyl taurine 0.3169 0.0015 5785.9 390.3 glutarylcarnitine (C5) 0.3149 0.0016 1565 276.1 benzoate 0.3109 0.0018 1291.5 179 threonine 0.3086 0.002 713 120.1 stearamide 0.3069 0.0021 2071 131 X - 16009 0.3068 0.0021 2191 222 N2,N2-dimethylguanosine −0.3065 0.0021 2157 312.1 1-linoleoylglycerophosphocholine 0.3036 0.0024 5574 520.4 dihydrocholesterol 0.2989 0.0028 2320.8 215.2 X - 11412 0.2977 0.0029 3836 204.2 1-stearoylglycerophosphocholine 0.2934 0.0034 5844 524.4 X - 17147 0.2882 0.004 4830 187.2 glycerol 0.2845 0.0045 1311 205 X - 12435 0.2824 0.0048 3174 357.2 glycerate 0.2818 0.0049 1360.7 189 cysteine-glutathione disulfide −0.2804 0.0052 821 427.1 heptanoate (7:0) 0.2792 0.0054 3695 129.2 X - 16945 −0.2783 0.0055 3457.9 351 4-vinylphenol sulfate −0.2775 0.0057 3323 199.1 1-docosahexaenoylglycerophosphocholine 0.277 0.0058 5518 568.4 N-palmitoyl taurine 0.2756 0.006 5556.8 362.4 dihomo-linoleate (20:2n6) 0.2755 0.006 5722 307.3 succinylcarnitine 0.2754 0.0061 1401 262.1 2-linoleoylglycerophosphocholine 0.2717 0.0068 5544 520.4 X - 13543 −0.2669 0.0079 1096 484.1 X - 11437 −0.2656 0.0082 2888 231 2-palmitoylglycerophosphocholine 0.2592 0.0099 5604 496.3

TABLE 7 Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values (if any) were imputed with the observed minimum for that particular compound). ID A B palmitoyl sphingomyelin 0.03 0.2 stearoyl sphingomyelin N/A N/A X - 13215 0.02 0.1 alpha-tocopherol 0.01 0.03 1-stearoylglycerol (1-monostearin) 0.38 1.21 2-ethylhexanoate 0.06 0.02 X - 16120 0.45 0.45 7-alpha-hydroxycholesterol 0.74 4.91 X - 17010 0.68 2.51 dimethylarginine (SDMA + ADMA) 0.29 0.63 threonate 0.61 0.22 S-propylcysteine 0.05 0.05 X - 12355 0.03 0.03 1-arachidonoylglycerophosphocholine 0.03 0.72 2-docosapentaenoylglycerophosphocholine 0.11 1.23 X - 11987 0.14 0.14 7-beta-hydroxycholesterol 0.91 1.81 1-palmitoylglycerol (1-monopalmitin) 0.87 1.52 X - 16940 0.12 0.12 caprylate (8:0) 0.1 0.12 X - 11549 0.13 0.13 1-docosapentaenoylglycerophosphocholine 0.22 1.12 caproate (6:0) 0.35 0.49 arachidonate (20:4n6) 0.43 4.48 X - 17612 0.21 0.3 3-hydroxybutyrate (BHBA) 0.18 0.34 2-arachidonoylglycerophosphocholine 0.05 1.4 2-stearoylglycerophosphocholine 0.02 0.2 pelargonate (9:0) 0.07 0.1 X - 15546 0.15 0.15 N-stearoyl taurine 2.12 1.18 glutarylcarnitine (C5) 0.27 0.27 benzoate 0.03 0.03 threonine 3.43 3.44 stearamide 0.99 1.06 X - 16009 0.2 0.2 N2,N2-dimethylguanosine 0.59 0.54 1-linoleoylglycerophosphocholine 0.31 0.93 dihydrocholesterol 0.23 0.74 X - 11412 0.13 0.21 1-stearoylglycerophosphocholine 0.01 0.45 X - 17147 0.07 0.08 glycerol 2.61 1.85 X - 12435 0.36 0.36 glycerate 3.38 9.94 cysteine-glutathione disulfide 0.04 0.04 heptanoate (7:0) 0.27 0.39 X - 16945 0.07 0.07 4-vinylphenol sulfate 0.02 0.02 1-docosahexaenoylglycerophosphocholine 0.07 0.88 N-palmitoyl taurine 1 0.94 dihomo-linoleate (20:2n6) 0.33 4.06 succinylcarnitine 0.32 0.32 2-linoleoylglycerophosphocholine 0.2 1.75 X - 13543 0.1 0.1 X - 11437 0.05 0.05 2-palmitoylglycerophosphocholine 0.08 0.9

TABLE 8 High-density lipoproteins in response to diet A and Diet B. Difference Diet A Diet B p- Parameter Mean StdDev Mean StdDev Mean StdErr value High-Density 170.7 28.9 209.6 24.4 −38.9 4.8 0 Lipoprotein

TABLE 9 Compounds validated by dietary change. Retention ID Index Mass palmitoyl sphingomyelin 2524 311.3 X - 13215 5179 149.2 alpha-tocopherol 2305.4 502.5 1-stearoylglycerol (1-monostearin) 2186.6 399.4 7-alpha-hydroxycholesterol 2300 456.2 X - 17010 3169.5 189 1-arachidonoylglycerophosphocholine 5554 544.3 2-docosapentaenoylglycerophosphocholine 5532 570.3 7-beta-hydroxycholesterol 2340 456.4 1-palmitoylglycerol (1-monopalmitin) 2119.5 371.3 1-docosapentaenoylglycerophosphocholine 5584 570.4 caproate (6:0) 2766 115.2 arachidonate (20:4n6) 5525 303.4 X - 17612 4298.5 567.4 3-hydroxybutyrate (BHBA) 1203.5 116.9 2-arachidonoylglycerophosphocholine 5524 544.3 pelargonate (9:0) 4847 157.2 stearamide 2071 131 N2,N2-dimethylguanosine 2157 312.1 1-linoleoylglycerophosphocholine 5574 520.4 dihydrocholesterol 2320.8 215.2 X - 11412 3836 204.2 1-stearoylglycerophosphocholine 5844 524.4 X - 17147 4830 187.2 glycerate 1360.7 189 heptanoate (7:0) 3695 129.2 1-docosahexaenoylglycerophosphocholine 5518 568.4 dihomo-linoleate (20:2n6) 5722 307.3 2-linoleoylglycerophosphocholine 5544 520.4 2-palmitoylglycerophosphocholine 5604 496.3

Example 3 (Low-Density Lipoprotein)

Metabolite correlations with low-density lipoproteins were determined based on plasma metabolomics (Table 10). This provided a predictive model of compounds which can influence low-density lipoproteins either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 11), and noting changes in low-density lipoproteins (Table 12), served as a validation model. The metabolite compositions of the two different diets were determined in order to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 13).

TABLE 10 Metabolite correlations with low-density lipoproteins. Correlations with a P value < 0.01 are reported. Correlation Retention ID Correlation P-Value Index Mass 1-stearoylglycerol (1-monostearin) 0.5795 4.04E−10 2186.6 399.4 X - 13215 0.5664 1.21E−09 5179 149.2 palmitoyl sphingomyelin 0.5397 9.79E−09 2524 311.3 alpha-tocopherol 0.4864 3.82E−07 2305.4 502.5 lysine 0.4624 1.64E−06 1836.7 317.2 lathosterol 0.4583 2.08E−06 2337 255.2 7-alpha-hydroxycholesterol 0.437 6.85E−06 2300 456.2 arabinose 0.4178 1.86E−05 1631.6 217 2-linoleoylglycerophosphoethanolamine 0.4106 2.68E−05 5650 476.4 glycerophosphorylcholine (GPC) 0.4041 3.68E−05 694 258.1 7-beta-hydroxycholesterol 0.4028 3.93E−05 2340 456.4 dimethylarginine (SDMA + ADMA) −0.3911 6.83E−05 812 203.2 1-linoleoylglycerophosphoethanolamine 0.3485 0.0004 5725 476.3 X - 12029 0.3474 0.0005 683 329.1 X - 12189 0.3425 0.0006 1249 273.1 X - 17010 0.334 0.0008 3169.5 189 2-palmitoylglycerophosphoethanolamine 0.3315 0.0009 5790 452.3 stearoyl sphingomyelin 0.327 0.001 #N/A #N/A 1-palmitoylglycerol (1-monopalmitin) 0.3154 0.0016 2119.5 371.3 caproate (6:0) 0.3147 0.0016 2766 115.2 N1-methyladenosine −0.3115 0.0018 #N/A #N/A 2-ethylhexanoate 0.3045 0.0023 1210.5 201.2 cystine 0.3045 0.0023 2015.3 145.9 X - 11987 −0.2896 0.0038 1409 139.2 5-methyl-2′-deoxycytidine −0.2866 0.0042 1480 242 threonine 0.284 0.0046 713 120.1 2-arachidonoylglycerophosphoethanolamine 0.2796 0.0053 5674 500.3 S-propylcysteine −0.2788 0.0054 2000.5 164.1 malate −0.2732 0.0065 1502 233 taurocholate 0.272 0.0067 5122 514.3 glycerol 2-phosphate 0.2683 0.0076 1691.8 243 glycerol 3-phosphate (G3P) 0.2648 0.0084 1719.7 357.1 X - 12355 −0.2621 0.0091 3559 206.1 2-oleoylglycerophosphoethanolamine 0.2601 0.0097 5848 478.3 N2,N2-dimethylguanosine −0.2598 0.0098 2157 312.1

TABLE 11 Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values (if any were imputed with the observed minimum for that particular compound). ID A B 1-stearoylglycerol (1-monostearin) 0.38 1.21 X - 13215 0.02 0.1 palmitoyl sphingomyelin 0.03 0.2 alpha-tocopherol 0.01 0.03 lysine 33.69 6.43 lathosterol 0.41 0.41 7-alpha-hydroxycholesterol 0.74 4.91 arabinose 10.21 2.48 2-linoleoylglycerophosphoethanolamine 0.56 2.58 glycerophosphorylcholine (GPC) 57.82 106.56 7-beta-hydroxycholesterol 0.91 1.81 dimethylarginine (SDMA + ADMA) 0.29 0.63 1-linoleoylglycerophosphoethanolamine 0.48 2.77 X - 12029 0.35 0.35 X - 12189 0.23 0.23 X - 17010 0.68 2.51 2-palmitoylglycerophosphoethanolamine 0.69 2.75 stearoyl sphingomyelin N/A N/A 1-palmitoylglycerol (1-monopalmitin) 0.87 1.52 caproate (6:0) 0.35 0.49 N1-methyladenosine N/A N/A 2-ethylhexanoate 0.06 0.02 cystine 0.37 0.37 X - 11987 0.14 0.14 5-methyl-2′-deoxycytidine 1.05 0.61 threonine 3.43 3.44 2-arachidonoylglycerophosphoethanolamine 0.33 10.33 S-propylcysteine 0.05 0.05 malate 9.52 4.85 taurocholate 6.67 0.61 glycerol 2-phosphate 3.63 4.01 glycerol 3-phosphate (G3P) 3.12 10.24 X - 12355 0.03 0.03 2-oleoylglycerophosphoethanolamine 0.43 6.03 N2,N2-dimethylguanosine 0.59 0.54

TABLE 12 Low-density lipoproteins in response to diet A and Diet B. Difference Diet A Diet B p- Parameter Mean StdDev Mean StdDev Mean StdErr value Low-Density 8.3 4.7 31.5 18.2 −23.2 3.6 0 Lipoproteins

TABLE 13 Compounds validated by dietary change. Retention ID Index Mass 1-stearoylglycerol (1-monostearin) 2186.6 399.4 X - 13215 5179 149.2 palmitoyl sphingomyelin 2524 311.3 alpha-tocopherol 2305.4 502.5 7-alpha-hydroxycholesterol 2300 456.2 2-linoleoylglycerophosphoethanolamine 5650 476.4 glycerophosphorylcholine (GPC) 694 258.1 7-beta-hydroxycholesterol 2340 456.4 1-linoleoylglycerophosphoethanolamine 5725 476.3 X - 17010 3169.5 189 2-palmitoylglycerophosphoethanolamine 5790 452.3 1-palmitoylglycerol (1-monopalmitin) 2119.5 371.3 caproate (6:0) 2766 115.2 5-methyl-2′-deoxycytidine 1480 242 2-arachidonoylglycerophosphoethanolamine 5674 500.3 malate 1502 233 glycerol 2-phosphate 1691.8 243 glycerol 3-phosphate (G3P) 1719.7 357.1 2-oleoylglycerophosphoethanolamine 5848 478.3 N2,N2-dimethylguanosine 2157 312.1

It should be understood that various changes and modifications to the presently embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims. 

The invention is claimed as follows:
 1. A pet food composition, comprising: protein, carbohydrates, fat, fiber, and at least 6 distinct metabolites including 2 distinct metabolites for modulating each one of cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal; wherein the metabolite that modulates cholesterol is selected from the group consisting of palmitoyl sphingomyelin, X-13215, 1-stearoylglycerol (1-monostearin), alpha-tocopherol, 7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 7-beta-hydroxycholesterol, X-17010, glycerophosphorylcholine (GPC), X-16120, 2-linoleoylglycerophosphoethanolamine, 5-methyl-2′-deoxycytidine, caproate (6:0), 1-linoleoylglycerophosphoethanolamine, glycerol 3-phosphate (G3P), 2-arachidonoylglycerophosphoethanolamine, glycerol 2-phosphate, dihydrocholesterol, pelargonate (9:0), stearamide, 2-palmitoylglycerophosphoethanolamine, malate, heptanoate (7:0), and mixtures thereof; wherein the metabolite that modulates high-density lipoproteins is selected from the group consisting of palmitoyl sphingomyelin, X-13215, alpha-tocopherol, 1-stearoylglycerol (1-monostearin), 7-alpha-hydroxycholesterol, X-17010, 1-arachidonoylglycerophosphocholine, 2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate (20:4n6), X-17612, 3-hydroxybutyrate (BHBA), 2-arachidonoylglycerophosphocholine, pelargonate (9:0), stearamide, N2,N2-dimethylguanosine, 1-linoleoylglycerophosphocholine, dihydrocholesterol, X-11412, 1-stearoylglycerophosphocholine, X-17147, glycerate, heptanoate (7:0), 1-docosahexaenoylglycerophosphocholine, dihomo-linoleate (20:2n6), 2-linoleoylglycerophosphocholine, 2-palmitoylglycerophosphocholine, and mixtures thereof; wherein the metabolite that modulates low-density lipoproteins is selected from the group consisting of 1-stearoylglycerol (1-monostearin), X-13215, palmitoyl sphingomyelin, alpha-tocopherol, 7-alpha-hydroxycholesterol, 2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC), 7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine, X-17010, 2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol (1-monopalmitin), caproate (6:0), 5-methyl-2′-deoxycytidine, 2-arachidonoylglycerophosphoethanolamine, malate, glycerol 2-phosphate, glycerol 3-phosphate (G3P), 2-oleoylglycerophosphoethanolamine, N2,N2-dimethylguanosine, and mixtures thereof; and wherein the pet food composition provides at least a 5% increase or at least a 5% decrease in the amount of at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in the companion animal upon administration of the pet food to the companion animal.
 2. The pet food composition of claim 1, wherein the composition comprises at least 9 distinct metabolites, including at least 3 distinct metabolites for modulating each one of cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal.
 3. The pet food composition of claim 1, wherein the composition comprises at least 12 distinct metabolites, including at least 4 distinct metabolites for modulating each one of cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal.
 4. The pet food composition of claim 1, wherein the metabolite that modulates cholesterol is selected from the group consisting of palmitoyl sphingomyelin, X-13215, 1-stearoylglycerol (1-monostearin), alpha-tocopherol, 7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 7-beta-hydroxycholesterol, X-17010, glycerophosphorylcholine (GPC), X-16120, 2-linoleoylglycerophosphoethanolamine, 5-methyl-2′-deoxycytidine, caproate (6:0), and mixtures thereof.
 5. The pet food composition of claim 1, wherein the metabolite that modulates high-density lipoprotein is selected from the group consisting of palmitoyl sphingomyelin, X-13215, alpha-tocopherol, 1-stearoylglycerol (1-monostearin), 7-alpha-hydroxycholesterol, X-17010, 1-arachidonoylglycerophosphocholine, 2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate (20:4n6), and mixtures thereof.
 6. The pet food composition of claim 1, wherein the metabolite that modulates low-density lipoprotein is selected from the group consisting of 1-stearoylglycerol (1-monostearin), X-13215, palmitoyl sphingomyelin, alpha-tocopherol, 7-alpha-hydroxycholesterol, 2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC), 7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine, X-17010, 2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol (1-monopalmitin), and mixtures thereof.
 7. The pet food composition of claim 1, wherein the pet food composition provides at least a 10% increase in the amount of high-density lipoprotein in the companion animal.
 8. The pet food composition of claim 1, wherein the pet food composition provides at least a 10% decrease in the amount of at least one of cholesterol and low-density lipoprotein in the companion animal.
 9. A method of modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in a companion animal, comprising: administering to the companion animal a pet food composition comprising protein, carbohydrates, fat, fiber, and at least three distinct metabolites for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins in the companion animal; wherein the metabolite that modulates cholesterol is selected from the group consisting of palmitoyl sphingomyelin, X-13215, 1-stearoylglycerol (1-monostearin), alpha-tocopherol, 7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 7-beta-hydroxycholesterol, X-17010, glycerophosphorylcholine (GPC), X-16120, 2-linoleoylglycerophosphoethanolamine, 5-methyl-2′-deoxycytidine, caproate (6:0), 1-linoleoylglycerophosphoethanolamine, glycerol 3-phosphate (G3P), 2-arachidonoylglycerophosphoethanolamine, glycerol 2-phosphate, dihydrocholesterol, pelargonate (9:0), stearamide, 2-palmitoylglycerophosphoethanolamine, malate, heptanoate (7:0), and mixtures thereof; wherein the metabolite that modulates high-density lipoproteins is selected from the group consisting of palmitoyl sphingomyelin, X-13215, alpha-tocopherol, 1-stearoylglycerol (1-monostearin), 7-alpha-hydroxycholesterol, X-17010, 1-arachidonoylglycerophosphocholine, 2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin), 1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate (20:4n6), X-17612, 3-hydroxybutyrate (BHBA), 2-arachidonoylglycerophosphocholine, pelargonate (9:0), stearamide, N2,N2-dimethylguanosine, 1-linoleoylglycerophosphocholine, dihydrocholesterol, X-11412, 1-stearoylglycerophosphocholine, X-17147, glycerate, heptanoate (7:0), 1-docosahexaenoylglycerophosphocholine, dihomo-linoleate (20:2n6), 2-linoleoylglycerophosphocholine, 2-palmitoylglycerophosphocholine, and mixtures thereof; and wherein the metabolite that modulates low-density lipoproteins is selected from the group consisting of 1-stearoylglycerol (1-monostearin), X-13215, palmitoyl sphingomyelin, alpha-tocopherol, 7-alpha-hydroxycholesterol, 2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC), 7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine, X-17010, 2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol (1-monopalmitin), caproate (6:0), 5-methyl-2′-deoxycytidine, 2-arachidonoylglycerophosphoethanolamine, malate, glycerol 2-phosphate, glycerol 3-phosphate (G3P), 2-oleoylglycerophosphoethanolamine, N2,N2-dimethylguanosine, and mixtures thereof.
 10. The method of claim 9, wherein the composition comprises at least four distinct metabolites for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins.
 11. The method of claim 9, wherein the composition comprises at least five distinct metabolites for modulating at least one of cholesterol, high-density lipoproteins, or low-density lipoproteins.
 12. The method of claim 9, wherein the method modulates at least two of cholesterol, high-density lipoproteins, and low-density lipoproteins and wherein the composition comprises at least six distinct metabolites, three distinct metabolites for modulating each of the at least two of cholesterol, high-density lipoproteins, and low-density lipoproteins.
 13. The method of claim 9, wherein the method modulates cholesterol, high-density lipoproteins, and low-density lipoproteins and wherein the composition comprises at least nine distinct metabolites, three distinct metabolites for modulating each of cholesterol, high-density lipoproteins, and low-density lipoproteins.
 14. The method of claim 9, wherein the administration is a regular administration and wherein the pet food composition provides at least a 5% increase or a 5% decrease in the amount of cholesterol, high-density lipoproteins, or low-density lipoproteins in the companion animal.
 15. The method of claim 9, wherein the modulating provides treatment of one of the following: hypercholesterolemia, hyperlipidemia, coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, deep vein thrombosis, inflammation, or pulmonary embolism. 